Project Summary Targeting Bcl-2 family of proteins for cancer drug discovery has recently been validated by the FDA approval of the Bcl-2 selective inhibitor, venetoclax, for the treatment of chronic lymphocytic leukemia. Given the well- documented importance of Bcl-xL in solid tumors and its contribution to drug resistance, strategies devised to minimize the on-target platelet toxicity associated with the inhibition of Bcl-xL could boost the therapeutic applications of drugs like ABT-263, a dual Bcl-2/Bcl-xL inhibitor, in cancer. We have been utilizing an emerging technology in drug discovery, known as Proteolysis Targeting Chimera (PROTAC), to design small-molecules that can recruit Bcl-2/Bcl-xL proteins to E ubiquitin ligases for induced degradation. Since these molecules, named as Bcl-PROTACs, rely on E3 ligases to induce protein degradation, it is possible for them to achieve cell/tissue selectivity if they target an E3 ligase that is differentially expressed in different cells or tissues. In addition, PROTACs could be more efficacious than their corresponding conventional inhibitors owing to their catalytic nature in inducing protein degradation and their ability to deplete protein levels rather than transiently block an active site. Therefore, PROTACs are more efficacious than traditional occupancy-driven protein inhibitors. Therefore, we hypothesize that PROTACs designed to recruit an E3 ligase that is minimally expressed in platelets for the targeted degradation of Bcl-2/Bcl-xL will have reduced platelet toxicity and improved antitumor activity compared with their corresponding Bcl-2/Bcl-xL inhibitors such as ABT-263. Our preliminary proof-of- concept studies have demonstrated the feasibility of this approach. To further test our hypothesis, we plan to pursue the following specific aims: 1) design and synthesize novel Bcl-PROTACs to further improve the therapeutic index; 2) determine if the Bcl-PROTACs have reduced platelet toxicity and enhanced antitumor potency compared with their corresponding Bcl-2/Bcl-xL inhibitors. Success in the proposed studies would represent a potential breakthrough in developing cancer therapies targeting Bcl-2 family proteins. The PROTACs, which post-transcriptionally knockdown Bcl-2/Bcl-xL proteins, would also be useful reagents for understanding Bcl-2/Bcl-xL functions.